Top-down analysis of immunoglobulin G isotypes 1 and

2 with electron transfer dissociation on a high-field

Orbitrap mass spectrometer.

Luca Fornelli1¶, Daniel Ayoub1, Konstantin Aizikov2, Xiaowen Liu3,4, Eugen

Damoc2, Pavel A. Pevzner5, Alexander Makarov2, Alain Beck6, and Yury O.

Tsybin1,7*

1 Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Fédérale de

Lausanne, 1015 Lausanne, Switzerland

2 Thermo Fisher Scientific GmbH, 28199 Bremen, Germany

3 Department of BioHealth Informatics, Indiana University-Purdue University

Indianapolis, 46202 Indianapolis, IN, USA

4 Center for Computational Biology and Bioinformatics, Indiana University

School of Medicine, 46202 Indianapolis, IN, USA

5 Department of Computer Science and Engineering, University of California in

San Diego, 92093 San Diego, CA, USA

6 Centre d’Immunologie Pierre Fabre, 74160 St Julien-en-Genevois, France

7 Spectroswiss Sàrl, EPFL Innovation Park, 1015 Lausanne, Switzerland

_________________________________________________________________________________ This is the author's manuscript of the article published in final edited form as:Fornelli, L., Ayoub, D., Aizikov, K., Liu, X., Damoc, E., Pevzner, P. A., … Tsybin, Y. O. (2017). Top-down analysis of immunoglobulin G isotypes 1 and 2 with electron transfer dissociation on a high-field Orbitrap mass spectrometer. Journal of Proteomics, 159, 67–76. https://doi.org/10.1016/j.jprot.2017.02.013

2

*Correspondence should be addressed to Dr. Yury O. Tsybin, Spectroswiss Sàrl,

EPFL Innovation Park, Building I, 1015 Lausanne, Switzerland. Email:

tsybin@spectroswiss.ch, phone +41 21 693 98 06

¶ Present address: Northwestern University, 2170 Campus Dr, 60208 Evanston,

IL, USA

Present address: Luxembourg Institute of Health, 1445 Strassen, Luxembourg

Running title: Top-down ETD of IgGs on Orbitrap Elite

List of abbreviation:

Automatic gain control, AGC

Electron transfer dissociation, ETD

Electron transfer dissociation – higher energy collisional dissociation, EThcD

Fourier transform mass spectrometry, FTMS

Keywords: electron transfer dissociation, ETD; immunoglobulin G, IgG;

Orbitrap, top-down

Manuscript date: June 29, 2017

3

(Abstract)

The increasing importance of immunoglobulins G (IgGs) as biotherapeutics calls

for improved structural characterization methods designed for these large (~150

kDa) macromolecules. Analysis workflows have to be rapid, robust, and require

minimal sample preparation. In a previous work we showed the potential of

Orbitrap Fourier transform mass spectrometry (FTMS) combined with electron

transfer dissociation (ETD) for the top-down investigation of an intact IgG1,

resulting in ~30% sequence coverage. Here, we describe a top-down analysis of

two IgGs1 (adalimumab and trastuzumab) and one IgG2 (panitumumab)

performed with ETD on a mass spectrometer equipped with a high-field Orbitrap

mass analyzer. For the IgGs1, sequence coverage comparable to the previous

results was achieved in a two-fold reduced number of summed transients, which

corresponds, taken together with the significantly increased spectra acquisition

rate, to ~six-fold improvement in analysis time. Furthermore, we studied the

influence of ion-ion interaction times on ETD product ions for IgGs1, and the

differences in fragmentation behavior between IgGs1 and IgG2, which present

structural differences. Overall, these results reinforce the hypothesis that gas

phase dissociation using both energy threshold-based and radical-driven ion

activations is directed to specific regions of the polypeptide chains mostly by the

location of disulfide bonds.

4

1. Introduction

Monoclonal antibodies (mAbs) are an effective therapeutic tool in a variety of

medical conditions such as cancer and inflammatory disease.[1] These large

glycoproteins constitute the fastest growing class of biotherapeutics.[2, 3] The

main class of antibodies employed as biopharmaceuticals is immunoglobulin G

(IgG). IgGs are large (~150 kDa) biomolecules with a quaternary structure made

of two ~25 kDa light and two ~50 kDa heavy chains, identical among themselves,

with the latter being N-glycosylated, Supporting Information Figure S1. The four

chains are interconnected and their tertiary/quaternary structure is reinforced

by inter- and intra-molecular disulfide bridges. Heavy chains can be translated

from different genes, and their sequence determines the so-called IgG isotype (or

subclass). In the pharmaceutical industry, three of the four IgG isotypes are of

interest: IgG1, IgG2, and IgG4. Although all isotypes share ~90% of homology in

their amino acid sequences, the structure of their hinge regions and the disulfide

bond connectivity differ significantly. IgG1 and IgG4 contain 16 disulfide bridges,

two in the hinge region linking the heavy chains, one disulfide bridge linking

each light chain to the corresponding heavy chain and 12 intramolecular bridges

(2 and 4 for each light and heavy chain, respectively). IgG2 contains 18 disulfide

bonds with four in the hinge region linking the two heavy chains together.[4, 5]

Figure S1 compares the structures of an IgG1 and an IgG2. Note that the C-

terminal cysteine of the light chain is linked to the third cysteine (from the N-

terminus) of the heavy chain in IgG2 (and also IgG4), whereas in IgG1 it is linked

to the fifth cysteine.

5

Due to their relevance as biotherapeutics, and with the raise of the so-

called biosimilars in the pharmaceutical market,[3, 6] high-throughput and

extensive characterization of the sequence and structure of IgGs is needed.[2]

Mass spectrometry (MS) plays a pivotal role in analysis of IgGs. MS-based

methods have been developed and implemented across all stages of mAb

production.[4] The basic workflow may include intact IgG mass measurement,

IgG fragment (reduced light and heavy chains or papain-produced fragments)

mass measurement (referred to as middle-up MS approach),[7] middle-down,[8,

9] extended bottom-up[10] and bottom-up MS[2, 11] approaches. Although

bottom-up approaches provide the most structural information today, they suffer

a number of drawbacks such as artifact introduction and lengthy sample

preparation.[12-14] Top-down mass spectrometry (TD MS)[15] refers to the gas-

phase dissociation of intact biomolecules and subsequent mass measurement of

the fragmentation products. Applied to IgGs, TD MS is a convenient way to obtain

primary structure information without the need of reducing the inter-chain

disulfide bonds in solution. Furthermore, this approach generally allows access

to sequence confirmation of terminal regions as well as variable domains. It also

allows the identification and localization of major modifications and the

characterization of major glycoforms.[16] It has the advantage of requiring a

highly simplified sample preparation and therefore minimal artifact introduction

induced by sample processing. Despite these advantages, TD MS is not yet

routinely used for the characterization of mAbs, primarily due to the large

number of highly charged products yielded by IgG fragmentation, which requires

6

high-resolution MS and the applications of techniques aimed at improving

sensitivity, such as spectral summation (averaging).[17]

Electron capture dissociation (ECD)[18, 19] and electron transfer

dissociation (ETD)[20] of whole proteins usually generate larger sequence

coverage than slow-heating activation methods (such as collision-induced

dissociation, CID).[21] ECD and ETD MS/MS are also known as powerful

methods for characterization of labile post-translational modifications, e.g.,

glycosylation.[22, 23] Fourier transform ion cyclotron resonance (FT-ICR) MS

provides the highest resolution and can be hyphenated with ECD, making it the

technique of choice for earlier TD MS experiments.[24-26] Notably, Mao et al.

achieved 25% sequence coverage of a purified IgG1 using qFT-ICR MS and ECD

when isolating a single charge state in a direct infusion ionization mode. They

increased the sequence coverage to ~33% with a broadband tandem mass

spectrometry (MS/MS), e.g., using all the IgG1 ions present in the charge state

envelope as precursors for ECD.[27] However, the general use of FT-ICR MS

instruments for IgG characterization is rare,[4] presumably due to their limited

presence in biopharmaceutical laboratories owing to the tedious maintenance

and operational costs, as well as oftentimes low efficiency of heavy precursor ion

transfer and capture in the ICR cells for subsequent ECD reaction.

With the advent of ETD efficiently performed in an external ion trap with

product ion detection in a high-resolution mass analyzer, e.g., Orbitrap

FTMS[28], FT-ICR MS,[29] or time-of-flight (TOF) MS,[30, 31] the performance of

TD MS has improved. Particularly, TD MS of proteins on-line separated with

7

reversed phase liquid chromatography (LC) prior to electrospray ionization (ESI)

became possible routinely.[32, 33] We have previously described the use of ETD

with a high-resolution qTOF MS reporting the overall sequence coverage of 21%

for mouse IgG1 and 15% for human IgG1 which provided structural information

on variable domains of both the heavy and the light chain.[34] As a result of a

more extensive investigation, we achieved 33% sequence coverage of a human

IgG1 using ETD with a hybrid linear ion trap-Orbitrap FTMS (considering c-, z-

and y-type product ions) equipped with a standard (moderate electric field, D30

type) Orbitrap mass analyzer (Orbitrap Velos Pro). The results obtained with

narrow (i.e., 100 m/z) and large (i.e., 600 m/z) isolation windows for precursor

ions were combined to increase the final number of identified product ions.[17]

In all the aforementioned TD MS studies, over one thousand (micro)scans (or

transients in a time-domain domain, in the case of FTMS) were summed

(averaged) to boost the sequence coverage.[17, 27, 34] Typically, several LC

MS/MS runs were acquired followed by data summation (averaging) to provide

the increased number of scans. The current state-of-the-art in TD MS, however,

does not enable the full sequencing of the disulfide bond protected domains of

light or heavy chain, neither it allows the direct localization of the glycosylation

site in the CH2 domain of the heavy chain. This is due to the compact protein

structure and its disulfide bond network (see Supporting Information, Figure

S1), which is believed to constitute the major limitation in IgG TD MS

characterization. Overall, the performance of all three approaches, using ETD on

8

a qTOF MS or on a linear trap-Orbitrap FTMS, as well as ECD on FT-ICR MS,

provide comparable results.

Here we investigated the potential of ETD implemented on a high-field

compact Orbitrap FTMS, namely the Orbitrap Elite,[35] for faster top-down

characterization of three therapeutic mAbs: adalimumab and trastuzumab (both

of the IgG1 subclass), and the IgG2 panitumumab. The high-field Orbitrap mass

analyzer is characterized by a higher frequency of ion oscillations along the

central electrode compared to the prior generation of Orbitrap mass

analyzers.[36, 37] Increased frequency of ion oscillations leads to the

corresponding enhancement of the resolving power over a fixed acquisition time.

Moreover, the implementation of a mixed absorption and magnitude mode FT of

spectral representation (known as enhanced FT, or eFT algorithm)[38] allows a

further gain (of up to two-fold) in acquisition rate at a constant resolution.[39] In

addition to the advantages of the employed mass analyzer, we optimized

precursor ion isolation and product ion transfer between the linear trap and the

Orbitrap to increase the efficiency of ETD-based TD MS. Finally, IgG TD MS was

performed under the optimized conditions with a two-stage ion activation

method, a combination of ETD and higher-energy collision dissociation

(HCD)[40] termed EThcD,[41] which has been recently applied to TD MS

experiments, demonstrating sequence complementarity with ETD.[42, 43]

9

2. Materials and methods

2.1 Chemicals. Water, acetonitrile (ACN), methanol (MeOH), formic acid (FA)

and trifluoroethanol (TFE) were obtained in LC-MS purity grade. Water, ACN and

MeOH were purchased from Fluka Analytical (Buchs, Switzerland). FA was

obtained from Merck (Zug, Switzerland), and TFE from Acros Organics (Geel,

Belgium).

2.2 Sample preparation. Therapeutic monoclonal antibodies of the IgG1 class,

namely adalimumab (Humira, Abbott Laboratories) and trastuzumab (Herceptin,

Genentech), as well as the IgG2 panitumumab (Vectibix, Amgen) are the

European Medicines Agency approved versions and formulations, available

commercially to the general public. Prior to direct infusion analysis, all

antibodies were buffer exchanged against a 150 mM ammonium acetate solution

pH 6.7 using Micro-Spin desalting columns (Zeba 75 µL, Thermo Fisher

Scientific, Zug, Switzerland).

2.3 Sample delivery and ionization. The antibodies were injected into the

mass spectrometer either by on-line coupled LC or by direct infusion in a positive

ion mode of ESI. In the former case, we employed an UltiMate 3000 LC system

(Thermo Fisher Scientific) equipped with a reversed phase C4 column (Hypersil

Gold, 1x100 mm, 5 µm particle size, Thermo Fisher Scientific, Runcorn, UK),

applying a gradient of ACN from 5 to 80% in 20 min, in presence of 0.1% FA, and

10

a flow rate of 100 µl/min generated by the loading pump. 1.5 µg of antibody (~10

pmol), diluted in 20% ACN and 0.1% FA, were typically loaded in each injection,

resulting in ~2 min elution of the IgG. The IonMax ion source (Thermo Fisher

Scientific) was operated at 3.7 kV, with sheath gas set to 20 and auxiliary gas to

10 arbitrary units. In the latter case, the antibody, buffer exchanged as described

above, was diluted in 50% ACN and 0.1% FA to a final concentration of ~10 µM;

6 µl of this IgG solution were loaded in a 20 µl loop connected to the injection

valve of the LC system and infused through a silica capillary to the nanoESI

source (Nanospray Flex ion source, Thermo Fisher Scientific) equipped with a

metallic emitter to which a 2.4 kV voltage was applied. The composition of the

solution used to push the IgG from the loop to the source was 39.9% water, 30%

ACN, 20% MeOH, 10% TFE, and 0.1% FA. The flow rate generated by the nano-

pump of the LC system was 0.6 µl/min.

2.4 Mass spectrometry. All experiments were performed on a hybrid dual linear

ion trap high-field Orbitrap FT mass spectrometer (Orbitrap Elite, Thermo

Scientific, Bremen, Germany) equipped with ETD and HCD. Similarly to our

previous report,[17] the S-lens RF level was set to 70% and the temperature of

heated transfer capillary was 350° C. Broadband mass spectra of intact IgGs and

tandem mass spectra of the same molecules were recorded in separate

experiments using ion detection in the Orbitrap FTMS (m/z 200-4000). All

Orbitrap FTMS scans were recorded averaging 10 microscans to improve the

SNR. Orbitrap FTMS was calibrated in the high mass range (m/z 2000-4000)

11

using ion at 2021.93954 m/z of commercial LTQ Velos ESI Positive Ion

Calibration Solution (Thermo Fisher Scientific). Intact mass measurements of

IgGs were performed at 15’000 resolution at 400 m/z with a target value of

charges (automatic gain control, AGC) of 1 million. For ETD experiments,

precursor ions were isolated between m/z 2400 and 2900 in the high pressure

LTQ and subsequently subjected to ETD reaction. The AGC value for

fluoranthene radical anions was set to 2 million charges, anion maximum

injection time was set to 50 ms and ETD duration (i.e., ion-ion interaction time)

was set either to 10 or 25 ms. Product ion detection in the Orbitrap mass analyzer

was performed with 120’000 resolution (at 400 m/z, eFT enabled) and an AGC

for precursor ions of 1 million charges.

For both intact mass measurements and MS/MS, the gas (N2) “delta

pressure” was of 0.1E-10 torr. The delta pressure corresponds to the difference

in the pressure measured in the so-called ultra-high vacuum region (i.e., UHV

region, or Orbitrap chamber) when nitrogen is injected into the HCD cell and

when the gas is completely shut down. A delta value of 0.1E-10 torr likely

corresponds to a pressure of ~1-2 mtorr of N2 in the HCD cell. Typically the delta

pressure for bottom-up proteomics (i.e., smaller size ions) is ~0.35E-10 torr.

Therefore, for TD MS the N2 gas pressure in the HCD cell is reduced compared to

the bottom-up proteomics. To further improve ion transmission to the high-

resolution mass analyzer from the linear trap, ions ejected from the linear trap

were first collisionally cooled in the HCD cell (a technique known as “HCD

12

trapping”), then transferred backward to the C-trap, and finally injected into the

Orbitrap as previously described.[44]

EThcD experiments were performed as follows. After ETD with 10 ms

duration, all ETD products, including both charge-reduced species and product

ions (precursors are generally completely consumed in ETD under the applied

conditions), were transferred from the linear trap to the HCD cell. Here an axial

potential was set to 25 or 40 V, so that instead of ion capture and relaxation a

second ion activation event could occur. Finally, ions were transferred back to

the C-trap and further into the Orbitrap for detection.

2.5 FTMS data processing and analysis. The data processing routine for

obtaining tandem mass spectra with an improved SNR was performed as

previously described.[17] Briefly, in both LC-MS/MS and direct infusion MS/MS

experiments, Orbitrap FTMS time-domain transients were recorded in MIDAS

.dat format[45] and underwent summation (averaging) before being processed for

time-to-frequency conversion. In this way it was possible to sum 384 ms

transients derived from separate LC runs or distinct direct infusion experiments.

Enhanced Fourier transform, eFT, or a standard magnitude mode Fourier

transform, mFT, was then applied to process transients and yield frequency-

domain spectra (internal processing at Thermo Fisher Scientific).[38] The thus

obtained frequency spectra were then converted into .RAW mass spectra (internal

processing at Thermo Fisher Scientific) for further viewing and processing with

XCalibur software (Thermo Fisher Scientific), as well as data analysis (vide infra).

13

Final ETD mass spectra were derived from summed transients recorded for 10

and 25 ms ETD duration experiments. For an in-depth evaluation of the effect of

ETD duration on IgG fragmentation, 10 and 25 ms ETD datasets were also

analyzed separately. The resolution gain of eFT versus mFT is known to be two-

fold.[38] However, to demonstrate the practically useful and presumably

sufficient resolution requirement for analysis of top-down ETD mass spectra of

IgGs, only the mFT-processed data are reported here. Results obtained with eFT

signal processing were found to be comparable to the mFT processing in terms

of protein sequence coverage (data not shown).

Previously, the combination of Xtract deconvolution software and ProSight

PC 3.0 product ion assignment software (both from Thermo Fisher Scientific) was

successfully employed for IgG TD MS data analysis.[30] Here, we extended this

approach by adding a workflow combining the MS-Deconv deconvolution

algorithm and MS-Align+ product ion assignment software (both developed at

University of California San Diego, CA, USA).[46] A comparison between the

results produced by MS-Deconv/MS-Align+ and by Xtract/ProSight PC is

presented in Figure S2, Supporting Information. Both the data analysis packages

led to comparable results. In greater detail, the MS-Deconv/MS-Align+ analyses

were realized according to the following steps: (i) ReAdW software was used to

convert .RAW files of ETD mass spectra into centroided mzXML files; (ii) a

customized version of MS Deconv was used to deconvolute the mzXML files using

a SNR cutoff of 3. The custom version of MS-Deconv was written to process the

data sets containing MS/MS information only, whereas the standard version of

14

MS-Deconv requires both MS and the directly corresponding to it MS/MS data

sets; (iii) MS-Align+ was used for product ion identification, considering c- and

z·- type ions for ETD experiments and c-, z·-, b- and y-type ions for EThcD data.

Cysteine residues were considered as not modified and all the mass tolerances

were set to 20 ppm. Manual validation was performed for cleavage sites identified

from product ions present only in a single charge state. The graphical

fragmentation maps reported herein were realized using either ProSightPC or

ProSight Lite software.[47]

2.6 Product ion abundance analysis. Abundances of identified product ions

were derived from MS-Align+ analysis. For each cleavage site, all the related

product ions were considered and grouped. Final abundances were obtained by

dividing the abundance values reported for each product ion over the ion charge

state, and summing all the resulting charge-normalized abundance values of

product ions generated from a single cleavage site. For plotting together product

ion abundance (PIA) analysis results of different sets of data, the abundances

were expressed as relative percentages of the most abundant product ion cluster

(i.e., the group of all the z-type ions, with different charge state, derived from a

single cleavage site) in each data set.

3. Results and discussion

3.1 Optimization of ETD MS/MS of IgG. To maximize ETD efficiency, we sought

to first optimize precursor cation selection and product ion transfer. In the

15

former work based on an Orbitrap Velos Pro mass spectrometer we isolated

adalimumab precursor ions around m/z 2750 (isolation window width: 100 m/z,

precursor ions from 53+ to 55+) or 2900 (isolation window width: 600 m/z,

precursor ions from 47+ to 57+). Considering the charge state envelope obtained

with the current LC-MS setting on the Orbitrap Elite for intact adalimumab, we

centered the isolation window at m/z 2700, with a width of 600 m/z, thus

including precursors from 50+ to 61+ (Figure S3, Supporting Information).

Higher charge state precursor ions are supposed to increase the fragmentation

efficiency in ETD and facilitate the separation of obtained product ions bound by

non-covalent bonds. Furthermore, cleavage preferences within protein structure

shift as a function of charge location. In regard to product ion transfer, we

applied a combination of reduction of the gas pressure in the HCD cell and the

“HCD trapping” technique, vide supra. The gas pressure reduction improves SNR

of large product ions by improving their transmission from LTQ into the HCD

cell and further within the Orbitrap analyzer. Trapping of ions in the HCD cell

(and not directly and only in the C-trap) allows the improved transmission also

of lighter ions, presumably counterbalancing the reduced damping effect in the

C-trap resulting from the lower gas pressure (Figure S4, Supporting

Information).

Overall, the implementation of these settings allowed for SNR increases in

the MS/MS mode for high m/z ions, which resulted in a reduction of the number

of transients that needed to be summed to obtain high-quality mass spectra in

comparison to our previous work, vide infra. Each ETD mass spectrum derived

16

from a combination of transients was obtained applying two different ion-ion

interaction times, 10 and 25 ms, Figure 1. These two ETD durations were chosen

to allow a direct comparison with our previous work, where we combined ETD

MS/MS data resulting from shorter and longer ion-ion interaction times, pre-

selected for fragmentation of intact denatured IgG precursor ions. Increasing

ETD duration enhanced the formation of product ions with a reduced charge

state, likely because of the occurrence of multiple electron transfer and

consequent charge reduction events. Nevertheless, the separate analysis of mass

spectra obtained with 10 or 25 ms ETD duration revealed that most of the

identified cleavage sites are shared between the two experiments (see product

ion abundance analysis results below), suggesting that ETD is primarily limited

by the retention of a compact conformation of the IgG in the gas phase. Summing

transients from these two experimental sets, though, increased the confidence

in the assignment of cleavage sites, as product ions obtained by the same

backbone cleavage are identified in multiple charge states.

3.2 ETD and EThcD MS/MS of intact human IgG1. The analysis of product

ions obtained by ETD MS/MS was performed for two IgG1 mAbs, adalimumab

and trastuzumab. Their sequences are 92% homologous, the differences being

almost entirely localized to the complementarity determining regions (CDRs)

involved in the antigen binding. Figure 2 A displays the ETD fragmentation map

of adalimumab, with the typical fragmentation pattern expected for this IgG

isotype. Extensive fragmentation is achieved on the heavy chain for the loop

17

interconnecting VH and CH1, the loop between CH2 and CH3 and part of the CH3

domain; on the light chain, the characterized region is represented by the loop

between VL and CL. For both chains, therefore, the sequenced areas are mainly

disulfide bond-free loops exposed to the solvent. The results of ETD MS/MS

application to trastuzumab are nearly identical to those for adalimumab (Figure

S5, Supporting Information). For both the IgGs only the CDR3 domains of light

and heavy chains are fully sequenced, and only a few product ions positioned on

CDR2 of heavy and light chain have been assigned for trastuzumab (which shows

slightly higher sequence coverage). As summarized in Table 1, 310 ETD MS/MS

scans (which corresponds to 3100 microscans or transients) were summed to

result in 23.8% and 26.5% sequence coverage for adalimumab and trastuzumab,

respectively. Notably, from the comparison of these results and the previously

reported ones it is apparent how, although the total sequence coverage (i.e., sum

of cleavages in light and heavy chains) is similar, the distribution of identified

cleavage sites between the two chains is changed, with the heavy chain being

more sequenced in the present report than in the previous one. For trastuzumab

we reached 29% sequence coverage which represents the highest value obtained

so far for the heavy chain of an intact IgG1 by ETD TD MS. On the other hand,

the light chain has been less sequenced with the new settings and data analysis

routine than in the past, but also for the drastically reduced number of scans

considered. Manual inspection of MS/MS spectra reveals that the missing

product ions are mainly those arising from cleavage sites located at the two

termini of the light chain: these are light, lowly charged ions that presumably

18

were not favorably transmitted with the newly employed settings (Figure S4,

Supporting Information). The fully sequenced region in the light chain,

corresponding to the loop between VL and CL, as mentioned above, remained the

same between the two studies, and it is equal between adalimumab and

trastuzumab (both of which often show complementary c- and z-type ions for the

same cleavage site). Cleavages outside this area do not follow any clear pattern

and seem randomly distributed.

To improve the sequence coverage of the IgG1 and, specifically, of the light

chain, we applied EThcD on adalimumab. We summed transients obtained with

EThcD (i.e., both product ions and charge-reduced species) performed at both

25 and 40 V. As shown in Figure 2B, EThcD produced fragmentation mainly in

the same areas previously covered by ETD, but also induced new cleavages at

the N-terminus of both light and heavy chain, and provided b- and y-type ions

from the N-terminal side of proline that could not be previously identified from

ETD data. The sequence coverage for the heavy chain of adalimumab is lower

than for ETD, but it passed from ~19% to ~28% for the light chain, as reported

in Table 1. Overall, the combination of 310 ETD scans and 260 EThcD scans,

for a total of 570 scans, produced 30.5% sequence coverage, which is the same

result obtained by averaging 1360 scans in our previous work based on the

Orbitrap Velos Pro. In addition to the reduced number of scans, a two-fold scan

speed improvement due to the implementation of the high-field Orbitrap mass

analyzer (higher frequency of ion axial oscillations) decreased the required

experimental time for mFT signal processing. Further two-fold improvement can

19

be achieved by the application of eFT signal processing. For instance, at 120’000

resolution (at 400 m/z, eFT signal processing) and under LC-MS/MS conditions,

120 transients (microscans) could be recorded per minute of IgG elution on the

Orbitrap Elite FTMS versus the previous 40 transients (microscans) on the

Orbitrap Velos Pro (mFT signal processing, 100’000 resolution at 400 m/z). The

ETD MS/MS experimental sequence, including cation accumulation, anion

injection and subsequent cation/anion interaction largely contributes to the

duty cycle, partially counterbalancing the time gain in FTMS data acquisition

due to combination of high-field Orbitrap and eFT.

Finally, from the comparison of the ETD TD MS results obtained with

different ion transfer parameters (especially the pressure conditions in the HCD

cell), those reported here and in the previous work, it seems beneficial to acquire

ETD TD MS data with both conditions and pool the data together. Indeed, results

reported in Figure S6 (Supporting Information) show that the sequence coverage

for ETD MS/MS of adalimumab with only c and z-ions is increased to 41.3% and

34.7% for light and heavy chain, respectively, with a total sequence coverage of

36.8%.

3.3 Product ion abundance analysis of ETD MS/MS of IgGs1. A PIA analysis

was conducted on ETD MS/MS data for the C-terminal portions of the almost

completely sequenced heavy chains of the investigated IgGs1. Figure 3 shows a

comparison between the relative abundances of z-ions from 10 ms ETD of

adalimumab, as well as 10 ms and 25 ms ETD of trastuzumab. Note that the

20

two IgGs share identical sequence in the analyzed area. For all three data sets,

the average abundance of product ions generated by the disulfide-free loop

connecting CH2 and CH3 is ~40% of the maximum, and global maxima are all

located there. Conversely, ions derived from the disulfide-protected area of CH3,

as well as those from the disulfide-free C-terminal end (which is, structurally

speaking, a part of the CH3 domain), generally have an abundance lower than

10% of the corresponding maximum. Furthermore, the global maxima for the

two 10 ms ETD data sets are both located at the cleavage site between K105 and

G106 (which results in z106 ions). Other local maxima correspond to the formation

of z96, z93, and z86 ions. The 25 ms data set shows a similar trend, with z106 still

abundant but surpassed by z93, z91, and z86. Therefore, the ETD results are

conditioned by the secondary and tertiary structures of the gaseous protein ions.

Moreover, similarly to ECD MS/MS,[48] the ETD radical process seems to be

affected by the electron transfer site, as the presence of basic residues at one

side of the cleavage maxima would suggest. Finally, the shift in maxima

occurring when passing from 10 to 25 ms ETD might be explained by the

increased probability of secondary electron transfer events when the ETD

duration is prolonged. The occurrence of secondary electron transfer and

consequent charge neutralization events might be confirmed by the reduced

average charge state of the product ions identified for 25 ms ETD in comparison

to 10 ms ETD experiments (Table S1, Supporting Information). The data suggest

that these events are most prominent at the flexible loop and with a CH3 domain

in a compact conformation. Therefore, despite the second fragmentation event,

21

the abundance of product ions originated within the sequence of the CH3 domain

is not higher than for the 10 ms experiments. Instead, we observe the shift

between z106 and z86 as maxima in the disulfide-free region. Visually, this is

represented in Figure 4 through a color-coded map based on crystal structure

of trastuzumab (image elaborated with The PyMol Molecular Graphics System,

version 1.3, Schrodinger, LLC). The passage from 10 ms to 25 ms ETD duration

induces the translation of highly frequent cleavage sites (represented in red)

towards the C-terminus, but still in solvent accessible positions. On the contrary,

the compact “immunoglobulin-like domain” corresponding to CH3, which

resembles the structure of a beta barrel, and contains an intra-molecular

disulfide bridge, remains poorly sequenced (and thus represented in black-blue).

These results reinforce the hypothesis that IgGs retain a relatively compact high-

order structure in the gas phase, even under denaturing ionization conditions.

3.4 ETD MS/MS of intact human IgG2. ETD MS/MS was performed on

panitumumab, an IgG2, to provide a confirmation to our hypothesis that

limitations in sequence coverage achieved for IgGs1 are mainly attributable to

gas-phase retention of a structured, compact conformation by the protein. As

displayed in Figure 5, identified product ions for this IgG are localized in the

areas sequenced in the experiments involving adalimumab and trastuzumab,

e.g., the solvent accessible loops interconnecting consecutive disulfide-protected

domains and the CH3 domain. In-depth analysis reveals that the C-terminal

portion of the heavy chain is highly sequenced, with 83 assigned z-type ions

22

versus 82 z-ions identified for trastuzumab, which has overall the highest

sequence coverage for its heavy chain (Table 1). Nevertheless, given that, in

accordance to what is observed for IgGs1, the hinge region is not sequenced also

in the case of panitumumab, differences between the two IgG isotypes are

present in the loop interconnecting VH and CH1. In the case of IgGs1, this loop

was fully sequenced, whereas for panitumumab only partial sequencing was

achieved. This can be explained by the presence in the loop of the IgG2 of Cys133,

involved in an inter-molecular disulfide bridge connecting the heavy with the

light chain. Notably, most of the identified c-type product ions are located to the

N-terminus of C133. Further studies are needed to determine whether ETD

cleavages were primarily impeded in the portion of the loop at the C-terminal

side of C133 or, conversely, cleavages occurred but primarily produced product

ions with portions of the light chain still present and were therefore not

identifiable by the present version of the data analysis approach. Finally,

panitumumab’s light chain has been sequenced in its central area (i.e., disulfide-

free loop between VL and CL), similarly to IgGs1, but its incomplete sequencing

and, importantly, the limited number of identified z-ions (nine, corresponding to

only ~50-30% of those assigned in the other experiments), could indicate that

the inter-molecular disulfide bridge with the heavy chain is less efficiently

cleaved in IgG2 than in IgG1.

23

4. Concluding remarks

Developments in Orbitrap FTMS technology mark a substantial improvement in

top-down MS performance. Experimentally, around three-fold faster acquisition

of IgG ETD data under optimized conditions in the Orbitrap Elite (high-field

compact Orbitrap analyzer) combined with a more than two-fold reduction in the

number of transients to be summed as required for a comprehensive top-down

analysis of IgGs resulted in about six-fold reduction of acquisition time in the

present study relative to the analysis with the Orbitrap Velos Pro. For the latter,

a standard trap with mFT yielded resolution of 100’000 at 400 m/z. Here,

resolution of 60’000 (at 400 m/z) was provided with mFT signal processing and

120’000 (at 400 m/z) when eFT algorithm was employed. The (moderate) level of

product ion spectral complexity generated in this top-down experiment, perhaps

due to a symmetric subunit structure of IgGs, demonstrated similar performance

in sequence coverage for mFT and eFT-generated mass spectra. The following

factors contributed to the reduction of number of scans needed for high quality

TD MS analysis: (i) selection of higher charged precursors, (ii) optimization of

transfer of large ETD product ions from the linear trap to the Orbitrap by

lowering the HCD cell gas pressure, (iii) combination of experiments conducted

with different ion-ion reaction times, and (iv) use of EThcD with two different

collisional energy values. Nevertheless, the previously determined limit in

sequence coverage, ~30%, could not be overcome. The primary cause for the

limited sequence coverage is the gas-phase retention of highly structured areas

in the IgG, mainly in correspondence of the immunoglobulin-like domains.

24

To facilitate user access to the described technology, algorithms and

methods for spectral summation across different experiments or/and for

transient acquisition and subsequent signal processing (absorption mode FT) of

summed transients where needed by spectral complexity have been made

commercially available (Spectroswiss, Lausanne, Switzerland). Further

extending the sequence coverage in TD MS of IgGs may be provided by an

improved ETD reaction cell,[49] chemical ionization source for front-end ETD on

Orbitrap FTMS,[50] and the use of ion activation methods alternative to ETD,

e.g., ultraviolet photodissociation.[51] Furthermore, improved sequence coverage

is achievable by combining ETD experimental data acquired with high HCD gas

pressure (i.e., maximizing light product ion transmission), and low gas pressure

(i.e., maximizing heavy product ion transmission), as it results from the

combination of present data with those acquired in standard operational

conditions with the Orbitrap Velos Pro. Finally, the probable presence of internal

products and product ions retaining parts of both light and heavy chains bonded

by inter-molecular disulfide bridges will require developments in the data

analysis tailoring this specific class of proteins.

5. Acknowledgements

We thank Kristina Srzentić and Anton Kozhinov for discussions and technical

support. We express our gratitude to Thermo Fisher Scientific for providing

access under license to Orbitrap Elite FTMS transient signals and related

functionality enabled by the developers’ kit. The work was supported by the

25

Swiss National Science Foundation (Projects 200021-125147 and 200021-

147006) and the European Research Council (ERC Starting grant 280271 to

YOT). Xiaowen Liu was supported by the National Institute of General Medical

Sciences, National Institutes of Health (NIH) through Grant R01GM118470.

26

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Figure captions

Figure 1. Top-down ETD Orbitrap FTMS of IgG1 (adalimumab) with ion-ion

interaction time of 10 and 25 ms. (A) Combined mass spectrum including

transients from both 10 and 25 ms measurements, with product ion population

arising from the former experiment highlighted in blue, and from the latter

indicated in red. (B) and (C) Expanded views of the two product ion populations,

with assigned product ions derived from the heavy chain. Note the difference in

the average charge state of the product ions of the two populations.

Figure 2. Fragmentation map of adalimumab by top-down Orbitrap FTMS. (A)

ETD MS/MS. (B) EThcD MS/MS. The heavy chain information is on top, the light

chain information is at the bottom.

Figure 3. Product ion abundance analysis of ETD top-down MS of adalimumab

with 10 ms ETD duration and trastuzumab with 10 and 25 ms duration. In the

case of trastuzumab, the color-coded histogram demonstrates the improvement

in sequence coverage obtained through the combination of 10 ms (magenta) and

25 ms (cyan) ETD MS/MS data. Whereas medium and large product ions (e.g.,

z117 and z63) are generally produced using 10 ms duration, smaller ions such as

z16, z17 and z18 are detected only in the case of a longer ETD duration, presumably

as the result of secondary fragmentation (i.e., re-fragmentation of larger product

ions).

35

Figure 4. Color-coded representation of ETD PIA results for the C-terminus of

the heavy chain of trastuzumab. (A) 10 ms ETD. (B) 25 ms ETD. The displayed

image is based on X-ray crystallography data (PDB entry: 3D6G).

Figure 5. Fragmentation map of panitumumab (IgG2) obtained with top-down

ETD Orbitrap FTMS.

Table 1. Sequence coverage obtained for different IgGs1 and IgG2 by top-down

ETD Orbitrap FTMS. Indicated scans consist of 10 microscans each. Note, only

c- and z-type ions were considered for ETD MS (for both “high-field” and

“standard” Orbitrap data), whereas also b- and y-type ions were included in

EThcD MS analysis.

Table 1.

Number of 

scans

N‐terminal 

product ions

C‐terminal 

product ions

Unique 

cleavage sites

Sequence 

coverage

N‐terminal 

product ions

C‐terminal 

product ions

Unique 

cleavage sites

Sequence 

coverage

high‐field Orbitrap

Adalimumab ETD 310 34 19 41 19.2% 39 78 117 26.0% 23.8%

Adalimumab EThcD 260 45 29 59 27.7% 22 58 80 17.8% 21.0%

Adalimumab combined 570 50 32 67 31.5% 53 82 135 30.0% 30.5%

Trastuzumab ETD 310 37 19 45 21.1% 55 75 130 29.0% 26.5%

Panitumumab ETD 300 34 9 34 15.9% 23 83 106 23.9% 21.3%

standard Orbitrap

Adalimumab combined 1360 54 59 84 39.4% 60 79 119 26.4% 30.6%

Light chain Heavy chainTotal sequence 

coverage

36

Figure 1.

37

Figure 2.

38

Figure 3.

39

Figure 4.

40

Figure 5.